Lipoprotein lipase (LPL), synthesized primarily in adipocytes, heart and skeletal muscle, plays a central role in the generation of free fatty acids utilized by these tissues for re-esterification and storage of triglycerides or as sources of energy. The expression of LPL in heart and adipocytes is highly regulated at both transcriptional and post-transcriptional levels. In order to identify regulatory elements which modulate the expression of LPL in adipocytes, we synthesized an expression vector in which a 5' flanking 734 bp fragment of LPL controls the expression of the luciferase reporter gene. Transfection of plasmids containing deletions in the 5' flanking region of LPL in 3T3-Ll adipocytes and HepG2 cells identified positive (-368 to -35) and negative (-724 to - 565)cis-acting regulatory elements that modulate LPL expression. Analysis of the proximal positive element by gel shift mobility assay and DNAse footprinting using 3T3-Ll nuclear extracts revealed specific binding to an octamer motif at position -46 relative to the transcriptional start site of LPL. Analysis with antibodies generated against the transcription factors, Oct-1 and Oct-2, indicate that this binding protein is Oct-1. Deletion of the octamer motif from different LPL promoter constructs resulted in a 75% reduction of normal transcriptional activity from both cell lines. Thus, by using deletion mapping analysis we have demonstrated the presence of specific elements within the 5' flanking region of LPL that modulate the expression of the enzyme in 3T3-Ll adipocytes. In addition, an octamer binding protein (Oct-1) present in 3T3-Ll nuclear extracts which binds specifically to the proximal positive regulatory element has been identified. We have demonstrated that Oct-1 is necessary for expression of LPL in 3T3-Ll adipocytes.